Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full.
Cells within a multicellular organism are connected by cytoplasmic bridges, which are termed plasmodesmata in plants (Lucas W J et al., 2009) and tunneling nanotubes in animals (Rustom A et al., 2004). Plasmodesmata were shown to actively and passively regulate intercellular trafficking of viral proteins, transcription factors, phloem proteins, mRNA and sRNA in plants (Lucas W J et al., 2009; Molnar A et al., 2010). An important recent development was the demonstration of the exchange of genetic material between cells in plant tissue grafts (Stegemann S & Bock R, 2009). However, there is no report yet on the intercellular movement of DNA-containing organelles, plastids and mitochondria, between plant cells.
During the past few years, supracellularity has emerged as a trait common to all life. Once thought to be a feature unique to plants, the physical continuity of cytoplasm and plasma membranes between neighboring cells has been observed in animal cells (Rustom A et al., 2004). These tunneling nanotubes were shown to be the conduits of active transport of organelles and cytoplasmic molecules between cells. Particularly relevant for this work, is the direct observation of transport of mitochondria through tunneling nanotubes in animal cells (Koyanagi M et al., 2005; Acquistapace A et al., 2011). Tunneling nanotubes and filopodia-like cytoplasmic bridges have also been observed linking unrelated bacterial cells and therefore may represent a universal mechanism for cellular communication and interdependence (Dubey G P & Ben-Yehuda S, 2011). Modulation of this process would represent an advance in the art in the creation of transplastomic plants.
Because male sterile maternal parental plants avoid the requirement for hand emasculation, such plants are highly desirable in hybrid seed production. Male sterility can either be caused by mitochondrial genes or by nuclear genes alone; the resulting conditions are known as cytoplasmic male sterility (CMS) and genetic male sterility (GMS), respectively. CMS is known to be associated with mitochondrial DNA sequences which have multiple rearrangements giving rise to chimeric mitochondrial genes. The CMS maternal parent is female fertile and produces hybrid seed upon pollination by the pollen of the paternal parent. Fertility of the CMS parent is restored when a restorer gene is incorporated in the nuclear genome. CMS-causing mitochondrial genes and nuclear restorer genes have been extensively reviewed in different crop systems (Carlsson et al., 2008; Chase, 2007; Chen and Liu, 2013; Gillman et al., 2009).
Cultivated tomato, Solanum lycopersicum (also known as Lycopersicon esculentum and/or Lycopersicon lycopersicum) is a crop in which no CMS has been described. One approach for obtaining useful forms of CMS in tomato included protoplast fusion for introduction of Solanum acaule or Solanum tuberosum mitochondria into tomato cells (EP 03663819 A1; Priority date Oct. 8, 1988). The process comprises the steps of (A) fusing tomato protoplasts that contain inactivated cytoplasmic elements with Solanum protoplasts that contain inactivated nuclear elements, to obtain a plurality of fusion products; and (B) regenerating at least one fusion product of said plurality into a whole, male-sterile tomato plant.
Transgenic induction of mitochondrial DNA rearrangements for CMS was described in tomato by the manipulation of the Msh1 nuclear gene that appears to be involved in the suppression of illegitimate recombination in plant mitochondria. Suppression of Msh1 expression by RNAi resulted in reproducible mitochondrial DNA rearrangement and a condition of male sterility (Sandhu et al., 2007).
When chloroplast DNA moves from cell to cell over the graft junction, sequencing of the plastid genome of graft transfer events confirmed the presence of a complete, unmodified incoming ptDNA in the new host. In contrast, the mitochondrial DNA in the graft transmission plants was chimeric, consisting of segments of N. undulata mtDNA (from CMS Partner 1) and fertile mitochondrial DNA (from N. sylvestris). The plant mitochondrial DNA is present in different size sub genomic circles formed by recombination via repeated sequences (Kubo and Newton, 2008; Logan, 2007; Sugiyama et al., 2005). In somatic cells there may be more mitochondria than mitochondrial genomes and the mitochondria may contain less than a complete mitochondrial genome (Preuten et al., 2010). Plant mitochondria are known to undergo cycles of fusion (Sheahan et al., 2005). Thus, fertility- or sterility-controlling mitochondrial DNA may move from cell to cell protected in intact organelles or as naked DNA.
Transformation of mitochondria with naked DNA has not yet been accomplished in higher plants (Niazi et al., 2013) and U.S. Pat. No. 5,530,191 (1996) entitled “Method for producing cytoplasmic male sterility in plants and use thereof in production of hybrid seed” describes production of CMS plants by the engineering of the chloroplast genome. The patent literature claims hybrid tomato, but the seed in these patents is always obtained by conventional crossing, involving manual removal of anthers and hand pollination. Claims of hybrid tomato patents focus on flavor enhancement (PCT/US2012/041478) or the benefits of seedless tomato obtained by using parthenocarpic genes (PCT/NL2000/000380; EP19990201787; EP2010000012146; US 20130189419).